Methods and compositions for treatment of restenosis

ABSTRACT

The present invention provides sequences capable of inhibiting osteopontin (OPN) expression. In particular, the sequences provided herein are antisense osteopontin oligonucleotide sequences. The present invention further provides methods for treating restenosis using antisense osteopontin oligonucleotide sequences. In particular, methods for treating restenosis following vascular surgery (e.g., percutaneous transluminal coronary angioplasty (PCTA) and directional coronary atherectomy (DCA)) by using antisense osteopontin oligonucleotide sequences are provided.

This application claims the benefit of U.S. Provisional Application No.60/054,967, filed Aug. 7, 1997, and is a National Stage entry under 35U.S.C. 371 of International Patent Application PCT/US98/16569, filedAug. 7, 1998.

This work was made with Government support by the National Institutes ofHealth. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention provides sequences capable of inhibitingosteopontin (OPN) expression. In particular, the sequences providedherein are antisense osteopontin oligonucleotide sequences. The presentinvention further relates to methods for treating restenosis usingantisense osteopontin oligonucleotide sequences, and in particular, totreating restenosis following vascular surgery.

BACKGROUND OF THE INVENTION

Atherosclerosis (for review see Ross, R. (1993) Nature 362:801-809 andHajjar et al., (1995) Amer. Scientist 83:460-467) is the principal causeof heart attacks, stroke, gangrene and loss of function of extremities.It accounts for approximately 50% of all mortalities in the USA, Europeand Japan (Ross, R. (1993) Nature 362:801-809). The present therapeuticstrategies for severe atherosclerosis in coronary arteries rely onangioplasty procedures (e.g., percutaneous trans-luminal coronaryangioplasty (PTCA), directional coronary atherectomy (DCA) or relatedangioplasty procedures), and coronary artery bypass surgery. Forexample, PTCA is the primary treatment modality in many patients withcoronary heart disease. PTCA can relieve myocardial ischemia in patientswith coronary artery disease by reducing lumen obstruction and improvingcoronary bloodflow.

While the use of interventional procedures has grown rapidly,reocclusion (or restenosis) of arteries is a serious complication whichoccurs in 30-50% of patients undergoing various angioplasty procedureswithin 3 days to 3 months. Restenosis results in significant morbidityand mortality and frequently necessitates further interventions, such asrepeat angioplasty or coronary bypass surgery.

Although the processes responsible for restenosis are not completelyunderstood, restenosis has been suggested to occur. at least in part, asa result of local inflammation, thrombosis and smooth muscle cellmigration (Ferrell et al. (1992) Circulation 85:1630-1631) andproliferation (Austin et al. (1985) J. Am. Coll. Cardiol. 6:369-375;Giraldo et al. (1985) Arch. Pathol. Lab. Med. 109:173-175) within theintima of coronary arteries. To date, no post-angioplasty treatment hasproven effective in the prevention or treatment of restenosis.

Thus, there is a need for methods and compositions for preventing and/ortreating restenosis. Preferably, these methods and compositions arespecific in their effect, easy to administer, and are effective over ashort period of administration with minimal adverse side-effects.

SUMMARY OF THE INVENTION

The present invention discloses novel osteopontin antisense sequenceswhich are useful for the treatment and prevention of restenosis. Thepresent invention further discloses methods of diminishing osteopontinexpression in a subject capable of developing restenosis in a tissue,methods of treating restenosis in a subject suspected of being capableof developing restenosis in a tissue, methods of reducing osteopontinexpression in a subject undergoing angioplasty, methods of treatingrestenosis in a subject undergoing angioplasty, and methods of detectingrestenosis in a subject.

In particular, the invention provides an antisense sequence comprising anucleic acid sequence complementary to at least a portion of the humanosteopontin cDNA polynucleotide listed herein as SEQ ID NO:15. While itis not intended that the present invention be limited to any particularantisense sequence, in one preferred embodiment the antisense sequenceis selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In addition, though thepresent invention is not limited to a particular type of linkage, in amore preferred embodiment, the antisense sequence comprises one or morephosphorothioate linkages. In a yet more preferred embodiment, theantisense sequence is entrapped in a liposome.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and an antisensesequence comprising a nucleic acid sequence complementary to at least aportion of the polynucleotide of SEQ ID NO:15.

Further provided by the instant invention are methods of diminishingosteopontin expression, comprising: a) providing: i) a subject suspectedof being capable of developing restenosis in a tissue; and ii) anosteopontin antisense sequence complementary of at least a portion ofthe polynucleotide of SEQ ID NO:15; and b) administering an amount ofthe sequence to the subject under conditions such that the osteopontinexpression is diminished.

Without intending to limit the present invention to any particularsubject, in one embodiment, the subject is undergoing angioplasty. Alsowithout limiting the invention to a particular surgical method, in amore preferred embodiment, the angioplasty is selected from the groupconsisting of percutaneous trans-luminal coronary angioplasty anddirectional coronary atherectomy.

In an alternative embodiment, and without limiting the invention to aparticular type of tissue, the tissue is coronary vascular tissue. In apreferred embodiment, the coronary vascular tissue is arterial.

In yet another alternative embodiment, without intending to limit theinvention to a particular sequence, the osteopontin antisense sequenceis selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

Although it is not intended that the present invention be limited to aparticular method of administering, in a further alternative embodiment,the administering is parenteral. In a preferred embodiment, theparenteral administering is intraarterial (i.e., to the artery which issubjected to angioplasty). In yet a more preferred embodiment, theintraarterial administering is by using a catheter. In a particularlypreferred embodiment, the catheter is a double balloon catheter.

In yet another alternative embodiment, the osteopontin antisensesequence is entrapped in a liposome.

The instant invention further provides methods of treating restenosis,comprising: a) providing: i) a subject suspected of being capable ofdeveloping restenosis in a tissue; and ii) an osteopontin antisensesequence complementary to at least a portion of the polynucleotide ofSEQ ID NO:15; and b) administering an amount of the sequence to thesubject under conditions such that the restenosis is diminished.

The present invention further provides methods of reducing osteopontinexpression in a subject undergoing angioplasty, comprising: a)providing: i) a subject undergoing angioplasty; and ii) an osteopontinantisense sequence complementary to at least a portion of thepolynucleotide of SEQ ID NO:15; and b) administering an amount of thesequence to the subject under conditions such that osteopontinexpression is diminished. In one embodiment, and without intending tolimit the invention to a particular sequence, the osteopontin antisensesequence is selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. In a preferredembodiment, the osteopontin antisense sequence comprises one or morephosphorothioate linkages. In a more preferred embodiment, theosteopontin antisense sequence is entrapped in a liposome. In yet a morepreferred embodiment, the administering is substantially contemporaneouswith the angioplasty. In a particularly preferred embodiment, theadministering is by using a catheter. In a most preferred embodiment,the catheter is a double balloon catheter.

The present invention also provides methods of treating restenosis in asubject undergoing angioplasty, comprising: a) providing: i) a subjectundergoing angioplasty; and ii) an osteopontin antisense sequencecomplementary of at least a portion of the polynucleotide of SEQ IDNO:15; and b) administering an amount of the sequence to the subjectunder conditions such that restenosis is diminished.

Also provided by the present invention are methods of detectingrestenosis in a first subject, comprising detecting a higher level ofosteopontin in a first tissue of a first subject suspected of beingcapable of developing restenosis in a second tissue relative to a levelof osteopontin in said first tissue of a second subject substantiallyfree of restenosis in said second tissue. In one embodiment, the firsttissue is selected from the group consisting of blood and plasma. In apreferred embodiment, the first tissue comprises monocytes comprisingthe osteopontin. In a more preferred embodiment, the second tissue iscoronary vascular tissue. In yet a more preferred embodiment, thecoronary vascular tissue is arterial.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the characterization of OPN expression in cultured CASMCs.FIG. 1, panel A, shows expression by RT-PCR (upper two panels), andWestern analysis (lower panel) in log-phase and confluent CASMCs. FIG.1, panel B, shows expression of OPN protein in semiconfluent CASMCs byimmunoprecipitation followed by Western blotting. FIG. 1, panel C, showsan autoradiogram of affinity cross-linked OPN in sub-receptor complex insubconfluent cultured CASMCs. FIG. 1, panel D, shows the results of anOPN binding study.

FIG. 2 shows the effect of OPN on CASMC (A) migration, (B) ECM-invasion,and (C) proliferation.

FIG. 3 shows (A) the detection of OPN-mRNA by in situ hybridization incoronary atherectomy arterial tissue (panels a and b), and in normalcoronary arteries (panels c and d); (B) the detection of OPN-mRNA byRT-PCR in normal coronary arteries (panel a), and in coronaryatherectomy arterial tissue (panel b); (C) the detection of OPN proteinby Western blot analysis in control (panel a) and coronary atherectomytissues (panel b).

FIG. 4 shows (A) Western blot analysis of OPN in plasma samples ofnormal controls (panel a) and atherectomy patients (panels b, c, and d);(B) densitometric analysis of plasma OPN bands from normal controls(C₁₋₃), atherectomy patients before DCA (P); and atherectomy patientsafter 1-4 weeks of DCA.

FIG. 5 shows the nucleotide sequence (SEQ ID NO:15) of a cDNA of humanosteopontin. This sequence contains a 5′ untranslated region of 67bases, followed by 942 bases encoding 314 amino acids, and 415 bases ofthe 3′ untranslated region.

FIG. 6 shows the effect of “LIPOFECTIN” alone (control), or in thepresence of OPN sense sequence SHOPN-P2 (SHOPN) or the antisensesequence ASHOP-P1 (ASHOPN) on the proliferation of human coronary arterysmooth muscle cells.

FIG. 7 shows the effect of transfection of CASMCs with “LIPOFECTIN”alone, or “LIPOFECTIN” containing different concentrations ofS-oligonucleotide antisense sequence ASHOPN-P1 (ASHOPN) (SEQ ID NO:9) onOPN-protein production as determined by immunoprecipitation followed byWestern blot analysis.

FIG. 8 shows the nucleotide sequence (SEQ ID NO:16) and deduced aminoacid sequence (SEQ ID NO:17) of rat osteopontin.

DEFINITIONS

The term “restenosis” refers to a recurrence of stenosis. The term“stenosis” as used herein refers to a narrowing of any canal in thecirculatory system including, but not limited to, valves (e.g., aorticstenosis which involves narrowing of the aortic valve orifice), coronaryarteries (e.g., coronary ostial sclerosis which involves narrowing ofthe mouths of the coronary arteries), carotid arteries, renal arteries,etc. Restenosis generally results from neointimal hyperplasia. The term“neointimal hyperplasia” refers to the development of a proliferativelesion in the intimal layer of a blood vessel. Neointimal hyperplasiaresults, for example, from migration of smooth muscle cells of thetunica media layer of the blood vessel toward the lumen into thesubintimal space below the endothelium (i.e., the inner lining of theblood vessel). These smooth muscle cells proliferate within the intimalspace and create a “mass effect” that narrows the vessel lumen andreduces oxygenation and nutritive blood flow.

The term “mRNA” as used herein refers to mature, processed mRNA or tounprocessed, nuclear pre-mRNA transcribed from a gene sequence.

The term “liposome” as used herein refers to a lipid-containing vesiclehaving a lipid bilayer as well as other lipid carrier particles whichcan entrap antisense oligonucleotides. Liposomes may be made of one ormore phospholipids, optionally including other materials such assterols. Suitable phospholipids include phosphatidyl cholines,phosphatidyl serines, and many others that are well known in the art.Liposomes can be unilamellar, multilamellar or have an undefinedlamellar structure.

The terms “entrap” and “incorporate” when made in reference to anoligonucleotide in a liposome are used herein to mean that theoligoncucleotide is at least partially contained somewhere within thewall of the liposome. Thus, an oligonucleotide entrapped in a liposomerefers to the presence of the oligonucleotide either partially orcompletely within the lipid vesicle or within a wall of the lipidvesicle. The molar ratio of lipids in the liposome to theoligonucleotide entrapped in the liposome is preferably between about100:1 and about 10,000:1, more preferably between about 500:1 and about5,000:1, and most preferably about 1,000:1.

As used herein, the term “therapeutic amount” refers to that amount of acompound required to reduce, delay, or eliminate undesirable pathologiceffects in a subject. A “therapeutic amount” of a compound when made inreference to restenosis refers to that amount of the compound whichwould diminish restenosis.

The term to “diminish restenosis” as used herein in reference to theeffect of a particular composition or of a particular method is meant toreduce, delay, or eliminate restenosis as compared to the level ofrestenosis observed in the absence of treatment with the particularcomposition or method. As used herein, the term “reducing” restenosisrefers to decreasing the intimal thickening that results fromstimulation of smooth muscle cell proliferation. The term “delaying”restenosis refers to increasing the time period between removal of astenosis (e.g., by use of surgical procedures) and onset of visibleintimal hyperplasia (e.g., observed histologically or by angiographicexamination). The term “eliminating” restenosis refers to completely“reducing” intimal thickening and/or completely “delaying” intimalhyperplasia in a subject to an extent which makes it no longer necessaryto surgically intervene in order to re-establish a suitable blood flowthrough the vessel by surgical means (e.g., by repeating angioplasty,atherectomy, or coronary artery bypass surgery). The effects ofdiminishing restenosis in a human subject may be determined by methodsroutine to those skilled in the art including, but not limited to,angiography, ultrasonic evaluation, fluoroscopic imaging, fiber opticendoscopic examination or biopsy and histology. The effects ofdiminishing restenosis in a non-human animal subject may be determinedby, for example, methods described herein including a reduction in theintimal/media cross-sectional ratio as measured by light microscopy offormalin-fixed tissue.

The term “substantially free of restenosis” when used in reference to atissue of a subject refers to a subject in which clinical symptoms ofrestenosis in the tissue are substantially absent. Methods fordetermining substantial absence of clinical symptoms are known in theart. For example, the substantial absence of restenosis in coronaryarterial vessels may be determined, for example, by cardiaccatheterization and coronary angiograms which are capable of revealingthe absence or presence of restenotic lesions, as well as by a thalliumstress test which is capable of determining coronary blood flow that isindicative of occlusion by restenotic lesions.

The term to “diminish osteopontin expression” as used herein inreference to the effect of a particular composition or of a particularmethod on a tissue is meant to reduce the level of osteopontinexpression in that tissue to a quantity which is less than the quantityof osteopontin expression in a corresponding control tissue which is,for example, not treated with that composition or method. For example,in order to determine whether a composition diminishes osteopontinexpression in arterial atherectomy tissue from a subject, an arterialatherectomy tissue sample is removed from the subject, treated in thepresence or absence of the composition, and the level of osteopontinexpression measured in the arterial atherectomy tissue which had beentreated in the presence or absence of the composition. The detection ofa level of osteopontin expression in the arterial atherectomy tissuewhich had been treated with the composition that is lower than the levelof osteopontin expression in the arterial atherectomy tissue which hadnot been treated with the composition demonstrates that the compositiondiminishes osteopontin expression.

The term “higher levels of plasma osteopontin” when made in reference toa first subject suspected of being capable of developing restenosis in atissue refers to a quantity of plasma osteopontin in the first subjectwhich is greater than the quantity of plasma osteopontin in a secondsubject substantially free of restenosis in that tissue, preferably atleast twice as great as, more preferably at least five times as greatas, and most preferably at least ten times as great as the quantity inthe second subject as determined by, for example, Western blot analysisof osteopontin and immunofluorescence for detection of osteopontin asdescribed herein.

The term “antisense” as used herein refers to a deoxyribonucleotidesequence whose sequence of deoxyribonucleotide residues is in reverse 5′to 3′ orientation in relation to the sequence of deoxyribonucleotideresidues in a sense strand of a DNA duplex. A “sense strand” of a DNAduplex refers to a strand in a DNA duplex which is transcribed by a cellin its natural state into a “sense mRNA.” Sense mRNA generally isultimately translated into a polypeptide. Thus an “antisense” sequenceis a sequence having the same sequence as the non-coding strand in a DNAduplex. The term “antisense mRNA” refers to a ribonucleotide sequencewhose sequence is complementary to an “antisense” sequence.

The term “oligonucleotide analog” as used herein refers to anoligonucleotide which comprises non-naturally-occurring portions. Thus,an oligonucleotide analog may have one or more altered sugar moieties,inter-sugar linkages, or altered base units. Altered inter-sugarlinkages include, for example, substitution of the phosphodiester bondsof the oligonucleotide with sulfur-containing bonds, phosphorothioatebonds, alkyl phosphorothioate bonds, N-alkyl phosphoramidates,phosphorodithioates, alkyl phosphonates and short chain alkyl orcycloalkyl structures.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

As used herein, the terms “vector” and “vehicle” are usedinterchangeably in reference to nucleic acid molecules that transfer DNAsegment(s) from one cell to another.

The term “expression vector” or “expression cassette” as used hereinrefers to a recombinant DNA molecule containing a desired codingsequence and appropriate nucleic acid sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism. Nucleic acid sequences necessary for expression in prokaryotesusually include a promoter, an operator (optional), and a ribosomebinding site, often along with other sequences. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals.

The terms “in operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule which is expressed using arecombinant DNA molecule.

As used herein, the terms “complementary” or “complementarity” when usedin reference to polynucleotides refer to polynucleotides which arerelated by the base-pairing rules. For example, or the sequence5′-AGT-3′ is complementary to the sequence 5′-ACT-3′. Complementaritymay be “partial,” in which one or more nucleic acid bases in one strandis not matched according to the base pairing rules with a nucleic acidbase in another strand. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in methods which depend upon binding betweennucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology or completehomology (i.e., identity). A partially complementary sequence is onethat at least partially inhibits a completely complementary sequencefrom hybridizing to a target nucleic acid is referred to using thefunctional term “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe (i.e., anoligonucleotide which is capable of hybridizing to anotheroligonucleotide of interest) will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous sequence to atarget under conditions of low stringency. This is not to say thatconditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget which lacks even a partial degree of complementarity (e.g., lessthan about 30% identity); in the absence of non-specific binding theprobe will not hybridize to the second non-complementary target.

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides inlength is employed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

When used in reference to nucleic acid hybridization the art knows wellthat numerous equivalent conditions may be employed to comprise eitherlow or high stringency conditions; factors such as the length and nature(DNA, RNA, base composition) of the probe and nature of the target (DNA,RNA, base composition, present in solution or immobilized, etc.) and theconcentration of the salts and other components (e.g., the presence orabsence of formamide, dextran sulfate, polyethylene glycol) areconsidered and the hybridization solution may be varied to generateconditions of either low or high stringency hybridization differentfrom, but equivalent to, the above listed conditions.

The term “hybridization” as used herein includes “any process by which astrand of nucleic acid joins with a complementary strand through basepairing.” [Coombs J (1994) Dictionary of Biotechnology, Stockton Press,New York N.Y.].

The terms “hybridizable” and “capable of hybridizing” refer to theability of one strand of nucleic acid to join with a completely orpartially complementary strand via base pairing under high or lowstringency conditions.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. “Stringency” typically occurs in a rangefrom about T_(m) to about 20° C. to 25° C. below T_(m). As will beunderstood by those of skill in the art, a stringent hybridization canbe used to identify or detect identical polynucleotide sequences or toidentify or detect similar or related polynucleotide sequences. Under“stringent conditions” SEQ ID NO:15 or fragments thereof will hybridizeto its exact complement and closely related sequences.

The term “atherosclerosis” refers to a form of arteriosclerosis in whichdeposits of yellowish plaques (i.e., atheromas) containing cholesterol,lipoid material, and lipophages are formed within the intima and innermedia of large and medium-sized arteries.

The term “angioplasty” refers to surgery of blood vessels as exemplifiedby percutaneous transluminal coronary angioplasty (PTCA), wherein aballoon in a catheter is inflated to open the lumen of an artery blockedby atherosclerotic plaques to allow blood flow, and by directionalcoronary atherectomy (DCA), wherein an atherosclerotic plaque is removedfrom the lumen of a blocked artery.

DESCRIPTION OF THE INVENTION

The present invention provides sequences capable of inhibitingosteopontin (OPN) expression. More particularly, the sequences providedherein are antisense OPN sequences. Also provided by the invention aremethods for treating restenosis. The compositions and methods providedby this invention are useful in treating restenosis associated withtraumatic injury to vascular walls. In particular, the compositions andmethods provided herein are useful for treating restenosis followingvascular surgery, e.g., percutaneous transluminal coronary angioplasty(PCTA), directional coronary atherectomy (DCA), and the like. Moreover,the compositions described herein find utility in inhibiting osteopontinexpression in vitro and in in vivo animal model systems.

To facilitate understanding of the inventions provided herein, thedescription of the invention is divided into (a) antisense osteopontin,and (b) methods for treating restenosis.

A. Antisense Osteopontin Sequences

Osteopontin was first identified in 1979 (Senger et al. (1979) Cell16:885-893) as a transformation-related phosphoprotein and was laternamed osteopontin (OPN) (Franzen et al. (1985) Biochem. J. 232:715-724).It is a secreted non-collagenous, glycosylated phosphoprotein (Oldberget al. (1988) J. Biol. Chem. 263:19433-19436; Nemir et al. (1989) J.Biol. Chem. 264:18202-18208; Craig et al. (1989) Int. J. Cancer46:133-137; Denhardt et al. (1993) FASEB J. 7:1475-1482) which binds tocell surface integrins (Hynes (1992) Cell 69:11-25), a family ofhetero-dimeric glycoprotein subunits designated α and β. These integrinsact as cell surface receptors for many ligands, including OPN (Oldberget al. (1986) Proc. Natl. Acad. Sci. USA. 83:8819-8823). OPN geneexpression has been reported to be a distinctive feature of rat aorticsmooth muscle cells (Giachelli et al. (1991) Biochem. Biophys. Res.Commun. 177:867-873). Moreover, rat and bovine smooth muscle cell(SMC)-migration is promoted by OPN (Liaw et al. (1994) Circ. Res.74:214-224). It has also been demonstrated that high levels of OPN-mRNAand protein are detectable in the rat and human aorta, and carotidarteries during neointima formation (Ikeda et al. (1993) J. Clin.Invest. 92:2814-2820; Giachelli et al. (1993) J. Clin. Invest.92:1686-1696; Shanahan et al. (1994) J. Clin. Invest. 93:2393-2402; Liawet al. (1995) J. Clin. Invest. 95:713-724). OPN overexpression has beenshown to associate with rat arterial SMC proliferation (Gadeau et al.(1993) Arteriosclerosis & Thrombosis 13:120-125), and its levels havebeen reported to increase in atherosclerotic plaques and duringrestenosis which follows balloon angioplasty (see, Rodan (1994)“Osteopontin overview,” In “Annals New York Acad. Sci.” pp 1-5). Mostinterestingly, it has been demonstrated that subjecting cultured cellsto intermittent compressive force, similar to the forces which may beproduced by some angioplasty procedures, causes OPN overexpression(Kubota et al (1993) Archs. Oral Biol. 38:23-30).

Without intending to limit the invention to a particular theory, datapresented herein suggest that one mechanism which contributes torestenosis is the migration of coronary artery smooth muscle cells(CASMCs) to the site of injury caused by angioplasty and subsequentproliferation of migrated CASMCs.

Also without limiting the invention to any particular theory ormechanism, data from in vitro and in vivo investigations presentedherein suggest that there may be a cascade of events which lead to thedevelopment of restenosis after angioplasty and that OPN plays bothautocrine and paracrine receptor-mediated roles which critically affectthe biology of coronary artery smooth muscle cells (CASMCs). OPN hasbeen reported to have chemotactic properties (Liaw et al. (1994) Circ.Res. 74:214-224) and has been demonstrated to induce proliferation inrat aortic smooth muscle cells (SMCs) (Gadeau et al. (1993)Arteriosclerosis & Thrombosis 13:120-125). Thus, a likely scenario isthat the inflammatory stimulus generated by the trauma of angioplasty isthe triggering event which causes infiltration of monocytes andmacrophages into the vascular smooth muscle layer. Since activatedmonocytes and macrophages are known to secrete OPN, the secreted OPN maybind to α_(v)β₃ integrin on CASMCs, which may in turn respond byexpressing yet more OPN. Secreted OPN then interacts with CASMCs in anautocrine or paracrine fashion and promotes their migration towards theintima where the angioplasty-induced injury has occurred. These cellsthen invade the ECM and finally, proliferate to cause reocclusion.

It has been reported that vascular smooth muscle cells, when stimulatedwith vitronectin, undergo haptotaxis (Naito et al. (1991) Exp. Cell Res.194:154-156), a process in which the cells migrate towards an increasinggradient of a chemoattractant. More recently, Senger et al. (Senger etal. (1996) Am. J. Pathol. 149:293-305) have demonstrated that OPN andits GRGDS-containing thrombin cleavage fragment promote tumor andvascular endothelial cell haptotaxis respectively, via the α_(v)β₃integrin. Data presented herein demonstrate that plasma OPN levelsdramatically increase following treatment of patients with the DCAprocedure. This increase may create an increasing gradient of thisprotein from the media of the arterial wall (where the CASMCs arenormally located) to the lumen of the artery, where the highestconcentration of OPN may be found. This data, combined with furtherresults provided herein, which demonstrate the ability of CASMCs tomigrate towards a higher concentration of OPN, to invade ECM, and toproliferate in response to OPN may explain the role of OPN in arterialocclusion (i.e., restenosis) following DCA procedure. Without intendingto limit the invention to any theory, it is hypothesized that theestablishment of such an OPN gradient in vivo results in the migrationof CASMCs from their original location in the arterial media, invasionof the arterial ECM, and arrival at their destination in the intima. Itis further hypothesized that CASMCs arriving at the intima proliferatein response to OPN-stimulation, thus resulting in reocclusion.

While there may be other factors involved in the pathogenesis of thisdisease process, results presented herein demonstrates that OPN and itsα_(v)β₃ integrin receptor play an essential role not only in stimulatingthe migration and ECM-invasion but also of proliferation of CASMCs.

Importantly, data presented herein which demonstrate for the first timethat lipofection of CASMCs with OPN-antisensephosphorothioate-oligonucleotides results in a drastic inhibition ofCASMC proliferation has far reaching clinical significance, i.e., thatreocclusion of vessels following vascular trauma may be diminished byadministration of OPN-antisense oligonucleotide sequences.

The present invention provides antisense OPN sequences. In oneembodiment, the antisense OPN sequence of the invention is SEQ ID NO:9.In another embodiment, the antisense OPN sequence provided herein is SEQID NO:10. In yet another embodiment, the antisense OPN sequencedisclosed by the present invention is SEQ ID NO:11. In a furtherembodiment, the antisense OPN sequence is SEQ ID NO:12. In yet a furtherembodiment, the antisense OPN sequence of the invention in SEQ ID NO:13.

The antisense OPN sequences of the invention are not limited to theantisense OPN sequences provided herein. Any antisense sequence iscontemplated to be within the scope of this invention if it is capableof reducing the level of expression of OPN to a quantity which is lessthan the quantity of OPN expression in a corresponding control tissuewhich is (a) not treated with the antisense OPN sequence, (b) treatedwith a corresponding sense OPN sequence, or (c) treated with a nonsensesequence. The terms “reducing the level of expression of OPN,”“diminishing osteopontin expression” and grammatical equivalents thereofrefer to reducing the level of OPN expression to a quantity which ispreferably 30% less than the quantity in a corresponding control tissue,more preferably 90% less than the quantity in a corresponding controltissue, and most preferably is at the background level of, or isundetectable by, a Western blot analysis of OPN, immunofluorescence fordetection of OPN, reverse transcription polymerase chain (RT-PCR)reaction for detection of OPN mRNA, or by in situ hybridization fordetection of OPN mRNA as described herein. When a background level orundetectable level of OPN or of OPN mRNA is measured, this may indicatethat OPN is not expressed, and thus that OPN is ineffective. A reducedlevel of OPN need not, although it may, mean an absolute absence ofexpression of OPN. The invention does not require, and is not limitedto, antisense OPN sequences which eliminate expression of OPN.

Antisense osteopontin sequences capable of reducing the level ofosteopontin expression include, for example, sequences which are capableof hybridizing with at least a portion of SEQ ID NO:15 under highstringency or low stringency conditions as described herein.

1. Design

Antisense OPN sequences within the scope of this invention may bedesigned using approaches known in the art. In a preferred embodiment,the antisense OPN sequences are designed to be hybridizable to OPN mRNAencoded by the coding region of the OPN gene as shown in FIG. 5, (SEQ IDNO:15) (Kiefer et al. (1989) Nucleic Acids Res. 17:3306). Antisense OPNsequences which are designed to hybridize to OPN mRNA which is encodedby the OPN gene coding region interfere with the normal function of themRNA, e.g., translocation to the situs for protein translation, bindingto ribosomes, etc., thus resulting in reduced translation of OPN mRNA.

Alternatively, antisense OPN sequences may be designed to reducetranscription by hybridizing to upstream nontranslated sequences,thereby preventing promoter binding to transcription factors.

In a preferred embodiment, the antisense oligonucleotide sequences ofthe invention range in size from about 8 to about 100 nucleotideresidues. In yet a more preferred embodiment, the oligonucleotidesequences range in size from about 8 to about 30 nucleotide residues. Ina most preferred embodiment, the antisense OPN sequences have 20nucleotide residues.

However, the invention is not intended to be limited to the number ofnucleotide residues in the oligonucleotide sequence disclosed herein.Any oligonucleotide sequence which is capable of reducing expression ofOPN is contemplated to be within the scope of this invention. Forexample, oligonucleotide sequences may range in size from about 3nucleotide residues to the entire OPN cDNA sequence of FIG. 5. The artskilled know that the degree of sequence uniqueness decreases withdecreasing length, thereby reducing the specificity of theoligonucleotide for the OPN mRNA.

In a preferred embodiment, the antisense oligonucleotide sequences ofthe invention comprise an oligonucleotide analog. In yet a morepreferred embodiment, the oligonucleotide analog contains one or morephosphorothioate bonds.

However, the antisense oligonucleotides of the invention are not limitedto oligonucleotide analogs with phosphorothioate linkages. The inventionis contemplated to include within its scope any oligonucleotidesequences so long as it is capable of hybridizing under low stringencyor high stringency conditions to the target human OPN mRNA.Oligonucleotides which hybridize under high stringency conditions to thetarget human OPN mRNA are preferred since such oligonucleotides exhibithigh specificity for the human OPN mRNA. Oligonucleotides which arecontemplated to be within the scope of this invention include, forexample, the antisense oligonucleotide sequences of the invention maycomprise naturally occurring nucleotide residues as well as nucleotideanalogs. Nucleotide analogs may include, for example, nucleotideresidues which contain altered sugar moieties, altered inter-sugarlinkages (e.g., substitution of the phosphodiester bonds of theoligonucleotide with sulfur-containing bonds, phosphorothioate bonds,alkyl phosphorothioate bonds, N-alkyl phosphoramidates,phosphorodithioates, alkyl phosphonates and short chain alkyl orcycloalkyl structures), or altered base units. Oligonucleotide analogsare desirable, for example, to increase the stability of the antisenseoligonucleotide compositions under biologic conditions since naturalphosphodiester bonds are not resistant to nuclease hydrolysis.Oligonucleotide analogs may also be desirable to improve incorporationefficiency of the oligonucleotides into liposomes, to enhance theability of the compositions to penetrate into the cells where thenucleic acid sequence whose activity is to be modulated is located, inorder to reduce the amount of antisense oligonucleotide needed for atherapeutic effect thereby also reducing the cost and possible sideeffects of treatment.

2. Synthesis

Antisense OPN oligonucleotide sequences may be synthesized using any ofa number of methods known in the art, as well as using commerciallyavailable services (e.g., Genta, Inc.). Synthesis of antisenseoligonucleotides may be performed, for example, using a solid supportand commercially available DNA synthesizers. Alternatively, antisenseoligonucleotides may also be synthesized using standard phosphoramidatechemistry techniques. For example, it is known in the art that for thegeneration of phosphodiester linkages, the oxidation is mediated viaiodine, while for the synthesis of phosphorothioates, the oxidation ismediated with 3H-1,2-benzodithiole-3-one,1,-dioxide in acetonitrile forthe step-wise thioation of the phosphite linkages. The thioation step isfollowed by a capping step, cleavage from the solid support, andpurification on HPLC, e.g., on a PRP-1 column and gradient ofacetonitrile in triethylammonium acetate, pH 7.0.

C. Methods for Treating Restenosis

The present invention provides methods for the treatment of restenosis.In one embodiment, the methods of the invention comprise administering atherapeutic amount of an OPN antisense oligonucleotide to a subjectunder conditions such that restenosis symptoms are diminished. In apreferred embodiment, the restenosis sought to be alleviated by themethods of the invention is that which may follow vascular trauma fromvascular surgical procedures such as angioplasty. Angioplasty may beperformed, for example, by percutaneous trans-luminal coronaryangioplasty (PTCA) or by directional coronary atherectomy (DCA). PTCAgenerally involves inserting a catheter (i.e., a plastic tube) with aballoon on the end into the blood vessel and inflating the balloon tohigh pressures to dilate the lumen of a blood vessel that is narrowede.g. by atherosclerosis (i.e., hardening of the artery). DCA involvesinserting a catheter with a probe at the end (the probe is generallymetallic in order to permit X-ray visualization during the surgicalprocedure) into the blood vessel and removing atherosclerotic tissuefrom the lumen of the vessel with the probe.

However, the methods of the invention are not limited to vascular traumafrom angioplastic procedures. Any procedure which results in restenosisis contemplated to be within the scope of the invention. Such proceduresinclude, for example, atheroectomy, placement of a stent (e.g., in avessel), thrombectomy, and grafting. Atheroectomy may be performed, forexample, by surgical excision, ultrasound or laser treatment, or by highpressure fluid flow. Introduction of a stent generally involvesintroducing a wire-mesh cylinder within the lumen of a stenotic vesselto increase the lumen diameter and restore blood flow. Thrombectomy maybe performed by, for example, introducing into the vessel apneumatically operated catheter which is fitted with rotating blades atthe tip to remove the thrombus or clot. Grafting may be achieved, forexample, by vascular grafting using natural or synthetic materials, orby surgical anastomosis of vessels such as during organ grafting.

1. Delivery

In a preferred embodiment, the antisense sequences provided herein aredelivered as liposomal oligonucleotides. In a more preferred embodiment,the liposomal composition used to entrap the antisense oligonucleotidesequences of the invention is “LIPOFECTIN.” “LIPOFECTIN” is a 1:1 (w/w)formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)and dioleoyl phosphatidylethanolamine (DOPE). The positively charged andneutral lipids from liposomes that can complex with nucleic acids.Successful direct physical transfer of genes into intact blood vesselsin vivo using cationic liposomes (e.g., “LIPOFECTIN”) has been reported[Nabel et al. (1990) Science 244:1285-1288]. Nabel et al. reported thatafter initial incubation with the liposomes using a double-ballooncatheter, expression of the transfected DNA could be detected in thevessel wall for up to five months. Lim et al. [Lim et al. (1991)Circulation 83:2007-2011] have also reported successfulcationic-lipid-mediated gene transfer into intact canine coronary andperipheral arteries. Successful lipofection of circulatory vessels hasalso been reported by Lynch et al. [Lynch et al. (1992) Proc. Natl.Acad. Sci. USA 89:1138-1142] and Flugelman et al. [Flugelman et al.(1992) Circulation 85:1110-1117].

The invention is not limited to the type or composition of the liposome.Any liposome which may be deemed useful by one of skill in the art foruse with the antisense molecules of the invention is contemplated to bewithin the scope of this invention. Liposomes of different compositionsare known in the art and are exemplified by those described herein orthose known in the art [e.g., U.S. Pat. No. 5,417,978 the contents ofwhich are herein incorporated by reference].

Delivery of the antisense oligonucleotides of the invention is notlimited to the use of liposomal oligonucleotides. The antisenseoligonucleotides provided herein may be delivered to a target cell invarious forms including, but not limited to, as free oligonucleotides oras oligonucleotides complexed with other compositions.

Where the antisense oligonucleotides of the invention are complexed withother compositions, such as with a combination of liposomes and theprotein coat of the inactivated hemagglutinating virus of Japan (HVJ)[Morishita et al. (1993) Proc. Natl. Acad. Sci. USA 90:8474-8478], orwith a combination of liposomes, inactivated HVJ coat protein andnuclear protein [Kaneda et al. (1989) Science 243:375-378; von der Leyenet al. (1994) FASEB J. 8:A802]. Antisense oligonucleotides complexedwith liposomes and the protein coat of HVJ have been shown to result ina more rapid cellular uptake and a 10-fold higher transfectionefficiency of antisense oligonucleotides or plasmid DNA than lipofectionor passive uptake methods [Morishita et al. (1993) J. Clin. Invest.91:2580-2585]. In an alternative embodiment, antisense oligonucleotidesequences may be administered in pluronic gels (BASF Wyandotte Corp.,Wyandottee, Mich.).

Transfer of antisense sequences into vascular smooth muscle cells may beaccomplished by other methods known in the art, includingre-implantation of cells modified in vitro [for a review, see, Dzau etal. (1993) Trends Biotechnol. 11:205-210].

Alternatively, antisense sequences may be introduced into a cell bytransferring into the target cell a vector capable of expression of theantisense sequence. In particular, viral-vector mediated gene transferis known in the art such as such as retrovirus, adenovirus,Hemagglutinating virus of Japan (HVJ; also referred to as Sendai virus).Vectors which express antisense OPN oligonucleotide sequences can floodcells with untranslatable antisense mRNA sequences which inhibitexpression of OPN either by inhibiting transcription of the OPN gene orinhibiting translation of an OPN-encoding mRNA. For example, vectorsderived from oncoretroviruses, such as the Moloney leukemia virus (MLV),integrate the transgene in the genome of the target cells withouttransferring any viral gene, two properties considered crucial for thesustained expression of the transgene. These retroviral vectors may beparticularly suitable for targeting OPN gene expression in proliferatinghuman smooth muscle cells since these vectors only transduce cells thatdivide shortly after infection [Miller et al. (1990) Mol. Cell. Biol.10:4239-4242], and do not transduce non-dividing cells [Naldini et al.(1996) Science 272:263-267].

Other virus-derived vectors which are suitable for in vivo gene transferare available in the art including human immunodeficiency virus-derivedvectors [Naldini et al. (1996) Science 272:263-267; Naldini et al.(1996) Proc. Natl. Acad. Sci. USA 93:11382-11388], adenovirus-derivedvectors [Lemarchand et al. (1993) Circ. Res. 72:1132-1138], retrovirusessuch as BAG and BAL [Wilson et al. (1989)Science 244:1344-1346]. Whileadenovirus-derived vectors are available, and their expression istemporary (i.e., making them suitable for treatment of acute diseasesuch as restenosis following angioplastic surgery) these vectors are notpreferred since an immune response is raised in vivo against thetransduced cells thus resulting in inflammation which would exacerbatethe risk of restenosis. Similarly, while vectors derived fromretroviruses (e.g., human immunodeficiency virus) are available, theiruse is not preferred as these vectors are expressed only in non-dividingcells, and would therefore be expected not to transduce proliferatingsmooth muscle cells during restenosis.

Methods for the design of a viral vector are known in the art.Generally, the design of a viral vector system relies upon thesegregation in the viral genome of cis-acting sequences involved in itstransfer to target cells from trans-acting sequences encoding the viralproteins. The prototype vector particle is assembled by viral proteinswhich are expressed from constructs stripped of all cis-actingsequences. These sequences are instead used to frame the expressioncassette for the transgene driven by an heterologous promoter. As theparticle will transfer only the latter construct, the infection processis limited to a single round without spreading. The safety andefficiency of an actual vector system depends on the extent to whichcomplete segregation of cis- and trans-acting functions is obtained.

2. Dosage

Those skilled in the art will recognize that the appropriate therapeuticdosage of the oligonucleotides of the invention for a given vascularsurgical procedure may be determined in in vitro and in vivo animalmodel systems, and in human preclinical trials. in vitro testing may beaccomplished using commercially available human coronary artery smoothmuscle cells (CASMCs) coupled with determination of the effect ofantisense oligonucleotide treatment on OPN expression as measured byWestern blot analysis, cellular proliferation and migration, and onextracellular matrix invasion, as described herein. In vivo testing of asuitable therapeutic dose may be accomplished using art-accepted animalmodels such as the rat carotid artery model described herein, in whichthe effect of antisense oligonucleotide treatment on OPN expression, DNAsynthesis and intimal/medial cross-sectional ratios are determined.

Generally, where the antisense oligonucleotides of the invention aredelivered as liposomal oligonucleotides, the dose of the antisenseoligonucleotide ranges preferably between about 1 μM and about 500 μM,more preferably between about 1 μM and about 100 μM, and most preferablybetween about 5 μM and about 15 μM. 3. Delivery Routes

In a preferred embodiment, the antisense oligonucleotides of the presentinvention are administered locally to the site of vascular trauma byusing an infusion catheter. Infusion catheters are commerciallyavailable (e.g., C. R. Bard Inc., Billerica, Mass.) and known in the art(e.g., infusion catheters described by Wolinsky in U.S. Pat. No.4,824,436, or by Spears in U.S. Pat. No. 4,512,762, the contents of bothpatents are herein incorporated by reference). The infusion catheter maybe conveniently a double balloon or quadruple balloon catheter with apermeable membrane.

The invention is not limited to local delivery by catheter. Theantisense oligonucleotides of the invention may be delivered to thesmooth muscle layers of a mammalian artery wall by a number of routessuch as, for example, the biodegradable materials exemplified by thosedescribed in U.S. Pat. No. 4,929,602 (the contents of which areincorporated by reference) which are impregnated with the sequences ofthe invention.

Alternatively, local delivery of the antisense nucleotides of theinvention may be achieved by using, for example, implanted osmoticpumps, or by inclusion of the oligonucleotide sequences into pluronicgels such as those available from BASF Wyandotte Corp., Wyandotte, Mich.One of skill in the art would appreciate that the antisense sequences ofthe present invention may need only to be delivered in a therapeuticdosage sufficient to expose the proximal (i.e., 6 to 9) cell layers ofthe intimal or tunica media cells which line the lumen of a bloodvessel. Such a dosage can be determined empirically by, for example,infusing vessels from suitable animal model systems and usingimmunohistochemical methods to detect the presence and cellularlocalization of OPN protein. Alternatively, dosage may also beempirically determined by conducting suitable in vitro investigation asdescribed herein.

It is further preferred, though not required, that the use of aninfusion catheter to administer the antisense sequences of the inventionbe performed substantially contemporaneously (i.e., during the samesurgical procedure which is employed to alleviate stenosis) with theperformance of the surgical procedures which result in vascular trauma.Such contemporaneity is desirable since it (a) is convenient, (b) avoidsunnecessary further trauma to the blood vessels which otherwise wouldresult from independent catheter infusion and angioplasty procedures,and (c) provides a greater probability of preventing restenosis sincesignificant elevation of the levels of circulating OPN occur as early as24 hours within performance of an angioplastic procedure as disclosed bythis invention, and since approximately 30-50% of the patientsundergoing angioplastic procedures suffer from restenosis within 3 daysto 3 months.

One of skill in the art would recognize that a suitable therapeuticdosages of antisense oligonucleotides administered in vivo by a catheteris dependent on several factors including, but not limited to, a) theatmospheric pressure applied during infusion; b) the time over which thecomposition administered resides at the vascular site; c) the nature ofthe employed composition which contains the oligonucleotides of theinvention; and/or d) the nature of the vascular trauma and therapydesired. Those skilled in the art will recognize that infiltration ofcompositions containing antisense oligonucleotide sequences into thesmooth muscle layers of a mammalian artery wall will probably bevariable and will need to be determined on an individual basis. Suchdetermination is routine, and follows similar principles as those knownto and applied by practitioners in the art in using a multitude of drugswhich are administered routinely.

While infusion catheters are contemplated to provide a preferredadministration route, one of skill in the art would recognize that othermethods for delivery or routes of administration may also be useful,e.g., injection by the intravenous, intralymphatic, intrathecal,intraarterial, or other intracavity routes. These routes are notpreferred since attaining a therapeutic level at the site of potentialrestenosis would require administration of a large amount ofoligonucleotide sequence which is costly

One of skill in the art knows that the sequences of the invention may beadministered using a number of pharmaceutically acceptable carriers(i.e., excipients). In a preferred embodiment, the pharmaceuticallyacceptable carrier is in liquid phase. Useful pharmaceuticallyacceptable carriers include generally employed carriers, such asphosphate buffered saline solution, water, emulsions (e.g., oil/waterand water/oil emulsions) and wetting agents of various types.

4. Timing and Number of Doses

It is contemplated that administration of antisense sequences of thepresent invention may be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic and otherfactors known to those skilled in the art.

The methods of the invention are not limited to the number or timing ofadministration of the antisense oligonucleotide sequences providedherein. For example, dosages for the prevention of restenosis followingangioplasty, may be applied prior to, simultaneously with, and/orsubsequent to the surgical intervention procedure. In one embodiment ofa dosing regimen, a “pre-loading” dose may be administered prior to orat the time of the intervention. A preloading dose may be a singlepre-loading dose (i.e., where the oligonucleotides of the invention areadministered at a single point in time) or a multiple pre-loading dose(i.e., where the oligonucleotides of the invention are administered atmultiple points in time). For example, a single pre-loading dose may beadministered about 24 hours prior to intervention, while multiplepre-loading doses may be administered daily for several days prior tointervention. In another embodiment of a dosing regimen, a“contemporaneous dose” may be administered, i.e. where theoligonucleotides of the invention are administered during the surgicalintervention procedure. In yet another embodiment of a dosing regimen, a“follow up” dose may be delivered subsequent to the interventionsurgical procedure. An example of a follow up dose is a dailyadministration one to two weeks or longer following intervention. One ofskill in the art would appreciate that the dosing regimen is selected soas to minimize the proliferative effect of osteopontin followingsurgical intervention for a time sufficient to substantially reduce therisk of, or to prevent, restenosis. One of skill in the art also knowsthat the dosing regimens may be determined empirically by in vitrotesting, in vivo testing in animal models, and by pre-clinical testingin human subjects. It is preferred, though not required, that a dosingregimen employ a contemporaneous dose.

The methods of the invention are not limited to the duration ofadministration of the antisense sequences provided herein.Administration may be for a short time (i.e., delivery over a period oftime equal to or less than about 2-3 minutes) or chronic (i.e.,continued or sporadic delivery which is continued over a period of timegreater than 10 minutes). Administration for a short time may be usefulto offset, at least partially, the strong stimulus for vascular smoothmuscle cell proliferation caused by highly traumatic injuries orprocedures such as angioplasty. On the other hand, chronic delivery of alower dose delivered to the traumatized site may provide furtherprotection against restenosis resulting from vascular smooth muscle cellproliferation in the traumatized area. In a preferred embodiment,administration is for a short time. More preferably, administration isfor about 2-3 minutes.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: CASMC (coronary artery smooth muscle cells); OPN(osteopontin); DCA (directional coronary atherectomy); DSS(disuccinimidyl suberate); PMSF (phenyl methyl sulfonyl-fluoride); PBS(phophate buffered saline); PMA (phosbol 12-myristate 13-acetate);RT-PCR (reverse transcription polymerase chain reaction); ECM(extracellular matrix); FCM (fibroblast conditioned medium); AmericanHistolabs (Rockville, Md.); BASF Wyandotte Corporation (Wyandotte,Mich.); Boehringer Mannheim (Indianapolis, Ind.); Charles Rivers(Michigan, OH); Chemicon (Temecula, Calif.); (Collaborative Research,Bedford, Mass.); Clonetics (San Diego, Calif.); Costar (Cambridge,Mass.); ICN (Biomedicals, CA), Pharmacia Biotecnology, Inc. (Piscataway,N.J.); Sigma (St. Louis, Mo.).

Example 1 Expression of OPN mRNA and Protein in Cultured Human CoronaryArtery Smooth Muscle Cells

The pattern of OPN-mRNA and protein expression in proliferating culturedhuman coronary artery smooth muscle cells (CASMCs) was investigatedusing reverse transcription polymerase chain reaction (RT-PCR) andWestern blot analysis on commercially available CASMCs. CASMCs(Clonetics) were cultured in smooth muscle cell basal medium (Clonetics)supplemented with insulin (5 μg/ml), human fibroblast growth factor (2ng/ml), human epidermal growth factor (0.5 ng/ml) and 5% fetal calfserum in a humidified atmosphere of 5% CO₂ and 95% air at 37° C.

Prior to the determination of mRNA and protein expression,immunofluorescence was used in order to determine whether these cellsare 100% smooth muscle cells using methods known in the art (Peri etal., (1994) DNA and Cell Biol. 13:495-503). Briefly, CASMCs during logphase of growth on microscopic slides were fixed in 4% bufferedparaformaldehyde, embedded in paraffin and histological sections wereprepared (American Histolabs). These cell samples were used forimmunofluorescent detection of (a) OPN using a previously characterizedrabbit OPN-antiserum (Chacklaparampil et al., (1996) Oncogene12:1457-1467), (b) SMC-specific α-actin using a monoclonal antibody(clone 1A4) to human SMC-specific α-actin (Sigma), and (c) α_(vβ) ₃integrin using mouse monoclonal antibody to human α_(v)β₃ (Chemicon).These cells expressed each of the three antigens (i.e., OPN,SMC-specific α-actin, and α_(v)β₃ integrin) thus confirming their smoothmuscle cell type.

A. Reverse Transcription Polymerase Chain Reaction

RNA from cultured CASMCs was extracted as previously described(Chomczynski et a. (1987) Anal. Biochem. 162:156159). Briefly, theCASMCs were grown in a 75 cm² flask, washed in ice-cold PBS three timesand lysed in 2 ml of RNA Zol B (Tel-Test, TX). The cell lysates (1 mleach) were transferred to microcentrifuge tubes and 100 μl of chloroformwere added to each tube. The upper aqueous layers were collected bycentrifugation at 12,000 rpm for 15 min. in a clinical centrifuge andthe contents (400 μl) transferred to another microcentrifuge tube. TheRNA samples were precipitated by adding 400 μl of isopropanol, collectedby centrifugation, washed with 75% cold ethanol and suspended in 15 μlDEPC-treated water. The concentration of RNA was measured by aspectophotometer.

The sequences of the primers and probe used for RT-PCR were derived fromthe sequence of human OPN cDNA shown in FIG. 5. The sequence of theantisense primer, hOPN-R (nt 928-909) was 5′-CTA CAA CCA GCA TAT CTTCA-3′ (SEQ ID NO:1) and of the sense primer, hOPN-L (nt 418-437) was5′-CAC CAG TCT GAT GAG TCT CA-3′ (SEQ ID NO:2). The PCR products weredetected by using a digoxigenin-labeled hOPN probe, i.e., hOPN-P₁ (nt647-628)=5′-TCC ATG TGT GAG GTG ATG TC-3′ (SEQ ID NO:3). Amplificationof the cDNA of a control house-keeping gene, i.e., glyceraldehyde3-phosphate dehydrogenase (GAPDH) was performed using the sense primer,GAPDH-L (nt 388-405) 5′-CCA TGG AGA AGG CTG GGG-3′ (SEQ ID NO:4) and theanti-sense primer, GAPDH-R (nt 582-563) 5′-CAA AGT TGT CAT GGA TGA CC-3′(SEQ ID NO:5). The probe, GAPDH-P (nt 549-531) was 5′-CTA AGC AGT TGGTGG TGC A-3′ (SEQ ID NO:6).

The results of RT-PCR are shown in FIG. 1A. Lane 1 contains CASMCs atlog phase of growth; Lane 2 contains CASMCs from confluent cultures. Theupper, middle and lower panels are OPN-mRNA, GAPDH mRNA, and OPN proteinrespectively. During log phase of growth these cells expressed elevatedlevels of OPN-mRNA (FIG. 1A, panel a: upper lane 1) compared to theconfluent cultures (FIG. 1A, panel a: upper lane 2). The GAPDH-mRNAsignals were identical (FIG. 1A, panel a: middle lanes 1 & 2) in bothnon-confluent and confluent cultures, demonstrating that thesedifferences were not due to variability in gel loading or degradation ofRNA during extraction.

The data in FIG. 1A panel a shows that OPN-mRNA and protein were easilydetectable when the cells were in log phase of growth while the levelwas significantly lower when the cells reached confluence.

B. Western Blot Analysis

The level of OPN in CASMCs was detected by Western blot analysis aspreviously described (Chacklaparampil et al., (1996) Oncogene12:1457-1467). Briefly, the specimens were homogenized in lysis buffer(50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl, 1% Nonidet P40, 15 μg/mlleupeptin and 0.5 μM PMSF), and centrifuged at 12,000×g for 10 min. Thesupernatants were electrophoresed on a 4-20% gradient SDS-polyacrylamidegel and electrotransferred to nitrocellulose membrane. The membraneswere blocked, incubated with rabbit anti-rat OPN antibody (previouslycharacterized by Chaklaparampil et al. (Chacklaparampil et al., (1996)Oncogene 12, 1457-1467) (1:250 dilution) and detected with ¹²⁵I-proteinA (ICN), followed by autoradiography.

The results of the Western blot analysis are shown in FIG. 1A panel a,lower panel. Western blot analysis of cell extracts showed that highlevels of OPN were expressed during the log phase of growth (FIG. 1A,panel a: lower lane 1), compared to confluent cultures (FIG. 1A, panela: lower lane 2).

C. Immunoprecipitation

An in vitro assay was used in order to determine whether an increase inOPN gene expression is detectable. Since phorbol myristate acetate (PMA)is known to induce OPN gene expression, CASMCs were stimulated with PMA,and OPN production was detected by immunoprecipitation of cell lysatesfollowed by Western blotting.

CASMCs were incubated with Phorbol 12-myristate 13-acetate (PMA) (250nM) at 37° C. for 24 h. The cells were immunoprecipitated using a kitaccording to manufacturer's (Boehringer Mannheim) instructions. Briefly,the cells were lysed with lysis buffer, centrifuged and the supernatantincubated with rabbit OPN-antibody for 1 h then with protein A-agaroseat 4° C. overnight. Bound complexes were pelleted by centrifugation,washed and electrophoresed. Western blot analysis was done as described,supra. The results are shown in FIG. 1, panel B.

In FIG. 1, panel B, left lane contains extracts from control cells whichwere not treated with PMA, while the right lane contains extracts fromcells stimulated with 250 nM PMA. The level of OPN protein inPMA-stimulated cells was markedly higher (FIG. 1, panel B: right lane)than that of unstimulated cells (FIG. 1, panel B: left lane). The twoOPN bands (right lane) detected upon PMA stimulation represent twoisoforms of this protein.

D. ¹²⁵I-OPN-Binding and Affinity-Crosslinking

Since OPN may exert its effect on CASMCs by interacting with itscell-surface receptor, the amount of membrane bound OPN was determinedusing ¹²⁵I-OPN-binding and affinity-crosslinking experiments.

For binding studies, purified hOPN was radioiodinated by theChloramine-T method (Hunter et al., (1962) Nature 194:495-496).Sub-confluent cultures of CASMCs were incubated with ¹²⁵I-OPN (3.3×10⁵cpm/well) in the absence or presence of varying concentrations ofunlabeled OPN in 0.5 ml Hank's balanced salt solution (HBSS), pH 7.6,containing 0.1% BSA. After incubation at 37° C. for 3 h, the reactionswere stopped by rapid removal of medium containing unbound radiolabeledOPN and the cells were washed and solubilized with 2 N NaOH. Theradioactivity was measured by gamma counter and the specific binding wascalculated by subtracting the non-specific binding from the totalbinding. The K_(d) value was determined by Scatchard analysis using“LIGAND” computer program (Munson et al., (1980) Anal. Biochem.107:220-239). ¹²⁵I-OPN was incubated with CASMC using increasingconcentrations of unlabeled OPN at 37° C. for 3 h. The results are shownin FIG. 1, panel D. The data were an average of duplicate experiments.

For affinity crosslinking experiments, sub-confluent CASMCs wereincubated with ¹²⁵I-OPN (6.6×10⁵ cpm/well) in 1 ml of HBSS, pH 7.6containing 0.1% BSA in the absence or presence of unlabeled OPN or GRGDSpeptide (1 μM) at 37° C. for 3 h. After washing, the cells wereincubated with 0.20 mM DSS in 1 ml HBSS, pH 7.6 at 37° C. for 30 min.The cells were scraped, collected by centrifugation and lysed in 40 μlof 1% Triton X-100 solution containing 1 mM PMSF, 20 μg/ml leupeptin and2 mM EDTA. The supernatants (30 μl) obtained by centrifugation wereelectrophoresed as described previously (Laemmli et al., (1970) Nature227:680-685) and autoradiographed. The results are shown in FIG. 1,panel C.

FIG. 1, panel C, shows the results of incubation of ¹²⁵I-OPN with CASMCin the absence or presence of unlabeled OPN or Gly-Arg-Gly-Asp-Ser(GRGDS) (SEQ ID NO:7) oligopeptide which corresponds to a portion of thecell adhesion sequence of OPN, and then crosslinked with disuccinimidylsuberate (DSS) (Pierce). These results show that the 300 kD protein banddisappeared when the cells were pretreated with either OPN or GRGDSpeptide. Furthermore, the 300 kD band was not detected in the absence ofDSS.

These results indicate that OPN binds to an approximately 300 kD cellsurface protein on CASMCs (FIG. 1, panel C) with high specificity andaffinity (Kd=1 nM) (FIG. 1, panel D). The results of immunoprecipitationwith α_(v)β₃ integrin antibody after binding and affinity-crosslinkingof ¹²⁵I-OPN with CASMCs established that the approximately 300 kDprotein band is indeed α_(v)β₃ integrin.

Example 2 Influence of OPN on In Vitro Coronary Smooth Muscle CellMigration, ECM-invasion, and Proliferation

In order to determine the effects of OPN on CASMCs, prior art-acceptedin vitro assay systems were used to evaluate the effects of this proteinon cellular migration, ECM-invasion and proliferation as follows.

A. CASMC Migration Assay

Migration of CASMC was performed using Transwell cell culture chamberswith an 8-μM pore size polycarbonate membrane (Costar) as describedpreviously (Yue et al., (1994) Exptl. Cell Res. 214:459-464). Briefly,sub-confluent human CASMC were trypsinized, centrifuged and resuspendedin basal medium (SmBM) supplemented with 0.2% BSA. This was followed bythe addition of 0.25 ml of cell suspension (5×10⁴ cells) to the uppercompartnent of the chamber. The lower compartment contained 0.5 ml ofbasal medium supplemented with 0.2% BSA together with 0.68 μg/ml OPN,1.36 μg/ml OPN, or buffer alone. After incubation at 37° C. for 24 h.,the non-migrated cells on the upper surface of the filters were scrapedand washed. The migrated cells were fixed in methanol, stained withGiemsa stain, counted under an inverted microscope and photomicrographed(120×) using a Zeiss photomicroscope (Axiovert 405 M). In separateexperiments, cells in the upper compartment were also treated withmonoclonal mouse anti-human α_(v)β₃-antibody (Chemicon) (10 μg/ml)before being assayed for migration in order to ascertain whether thisOPN-stimulated migration is mediated via α_(v)β₃. Preimmune IgGtreatment served as a non-specific control. The results are shown inFIG. 2A.

FIG. 2A shows that the rate of migration of CASMCs was enhanced withincreasing concentrations of OPN used as chemoattractant. Additionally,the OPN-induced migration was blocked when the cells were pre-treatedwith α_(v)β₃ integrin-antibody prior to performing each of these assays.A pre-immune IgG, used as a control, failed to exert any inhibitoryeffect on OPN-induced migration.

B. ECM-Invasion Assay

The ECM-invasion assay was performed using a commercially available24-well matrigel-coated invasion chamber (Collaborative Research) asdescribed previously (Kundu et al., (1996) Proc. Natl. Acad. Sci. (USA)93:2915-2919). Briefly, the confluent CASMC were trypsinized,centrifuged, and resuspended in basal medium supplemented with 0.1% BSA.The lower compartment of the invasion chamber was filled withfibroblast-conditioned medium (FCM) which served as a chemoattractant.The invasion assays were initiated by inoculating the upper chamber withcells (1×10⁵/well) which were either untreated or treated with varyingconcentrations of OPN (0.5-2.0 μg/ml). After incubating at 37° C. for 24h, the cells in the upper chamber were discarded, the matrigel wasscraped clear and the cells which had invaded the matrigel and migratedto the lower surface of the filter, were fixed, stained, counted andphotomicrographed (120×) as described above. The cells were alsopre-treated with mouse anti-human α_(v)β₃ antibody (Chemicon) (10 μg/ml)as described above to determine if the OPN-induced invasion was mediatedvia α_(v)β₃. Preimmune IgG was used as a non-specific control. Theresults are shown in FIG. 2B.

The results show that OPN-treatment of the cells enhanced theirinvasiveness (FIG. 2B) when tested on “MATRIGEL,” an artificial ECM, ina dose-dependent manner. The OPN-induced ECM-invasion was blocked whenthe cells were pre-treated with α_(v)β₃ integrin-antibody prior toperforming each of these assays. A pre-immune IgG, used as a control,failed to exert any inhibitory effect on OPN-induced ECM-invasion.

C. CASMC Proliferation Assay

Proliferation studies were carried out in the presence ofplatelet-derived growth factor-AB (PDGF-AB) as it has been suggestedthat in vivo platelet activation may contribute to the pathogenesis ofrestenosis. CASMCs were cultured as described above and the cells werestarved in serum free media for 48 h. The proliferation assays wereperformed as described previously (Monfardini et al., (1995) J. Biol.Chem. 270:6628-6638). Briefly, the cells were incubated in the absenceor presence of PDGF-AB (100 ng/ml) (Upstate Biotechnology, Lake Placid,N.Y.) and increasing concentrations of OPN (1.0-6.0 μg/ml) at 37° C. for24 h. In separate experiments, cells were pre-treated with either mouseanti human α_(v)β₃-antibody (5 μg/ml), preimmune IgG or GRGDS peptide(10 nM) followed by OPN (3.0 μg/ml). After 4 h, [3H]thymidine (1 μCi/ml)was added and the cells were maintained in culture for an additional 24h under the same culture conditions as described previously. Afterremoving the supernatants, the cells were washed with basal medium andlysed in 50% TCA. The acid precipitable cell-bound radioactivity wasmeasured using a scintillation counter (Beckman). The results are shownin FIG. 2C.

FIG. 2C shows that OPN-treatment of CASMCs also stimulated theirproliferation in a dose-dependent manner. While PDGF-AB alone hadvirtually no effect on CASMC proliferation, treatment of these cellswith OPN had a dramatic dose-dependent effect when used in conjunctionwith 100 ng/ml of PDGF-AB (FIG. 2C). Treatment of the cells with OPNalone yielded a modest proliferative response.

Interestingly, treatment of CASMCs with Gly-Arg-Gly-Asp-Ser (GRGDS)oligopeptide, or with α_(v)β₃ antibody drastically inhibited OPN-inducedproliferation (FIG. 2C).

Taken together, these results indicate that OPN gene expression isenhanced in proliferating, compared to contact-inhibited CASMCs, andthat treatment of these cells with purified OPN stimulated theirmotility, ECM-invasion, and proliferation. Moreover, these effects ofOPN are transduced via α_(v)β₃ integrin. Importantly, these data alsosuggest that OPN-antisense sequences may be useful for the inhibition ofOPN-mediated effects.

Example 3 OPN mRNA and Protein Expression in Human Coronary AtherectomyTissues

The above-discussed data obtained from in vitro investigations oncultured CASMCs raised the possibility that CASMCs which are located invivo on the arterial wall migrate from that location, invade the ECM,and proliferate to cause the occlusion which is associated with therestenosis observed following DCA.

Two questions, on which the prior art is silent, were particularlyimportant. The first was whether a distinction could be made betweenatherosclerotic and non-atherosclerotic coronary arterial tissues solelyon the basis of OPN-mRNA and protein expression patterns. If such wasthe case, the second question was whether the arterial tissues whichproduce OPN also express one of its receptors, the α_(v)β₃ integrin.These questions were addressed by the determination of the expression ofOPN-mRNA, OPN protein and α_(v)β₃ integrin protein in control andcoronary atherectomy tissues from human subjects.

A. Atherosclerotic Tissue Expresses α_(v)β₃ Integrin Protein and HigherLevels of OPN-mRNA and OPN Protein than Control Tissue

Coronary atherectomy tissues was obtained from 13 DCA-patients i.e.,patients who participated in an approved clinical research protocol andin whom directional coronary atherectomy (DCA) was clinically indicated.Informed consent was obtained from all patients in whomatherectomy/angioplasty was clinically indicated. Autopsy specimens ofnormal coronary arteries from 6 subjects (ages 18-68) at autopsy whodied of non-cardiac causes and had no evidence of atherosclerosis servedas controls. A summary of profiles of patient and control subjects ispresented in Table 1.

TABLE 1 Profile of Patients* and Controls** Number of Patients Age RangeSex 13 (DCA-patients) 43-62 2 F 11 M 6 (Controls) 18-68 2 F 4 M*Informed consent was obtained after the nature and possibleconsequences of the atherectomy procedure were explained. **No evidenceof coronary atherosclerosis at autopsy: death due to non-cardiac causes.

DCA-patient and control tissues were used to detect OPN-mRNA by in situhybridization and RT-PCR, α_(v)β₃ integrin protein byimmunofluorescence, and OPN protein by both immunofluorescence andWestern blotting. The atherectomy tissue samples, immediately afterremoval, were divided aseptically into three parts for RNA extraction,Western blot analysis and in situ hybridization, respectively.RNAse-free equipments and reagents were used for collection and storageof tissues used for in situ hybridization and RNA extraction. Controlsamples obtained at autopsy were prepared under the same conditions.

1. In situ Hybridization

For in situ hybridization, a digoxigenin-labeled oligonucleotide probederived from the sequence of human OPN cDNA (FIG. 5, SEQ ID NO:15) wasused as follows. Sections of paraformaldehyde-fixed tissues were placedon ribonuclease-free polylysine-treated glass slides (AmericanHistolabs, Inc.) and in situ hybridization was carried out as previouslydescribed [Peri et al. (1995) J. Clin. Invest. 96:343-353]. The hOPNprobe, hOPN-P₂ (nt 647-608): 5′-TCC ATG TGT GAG GTG ATG TCC TCG TCT GTAGCA TCA GGG T-3′) (SEQ ID NO:8), was 3′ end-labeled withdigoxigenin-11-ddUTP (Boehringer Mannheim) as described previously [Periet al. (1993) J. Clin. Invest. 92:2099-2109]. The slides werephotomicrographed with a Zeiss Axiomat photomicroscope (magnification400×). The results of in situ hybridization are shown in FIG. 3A.

FIG. 3A, panels a & b and c & d are bright field photomicrographs ofcoronary atherectomy and normal coronary artery tissues, respectively.In situ hybridization with an OPN probe showed that atherosclerotictissues obtained from DCA-patients expressed very high levels ofOPN-mRNA while it was virtually undetectable in control samples.

2. Reverse Transcription Polymerase Chain Reaction

For RT-PCR, reverse transcription of total RNAs from DCA-patients andcontrols and cDNA amplifications were performed according to the methoddescribed previously (Peri et al., (1993) J. Clin. Invest. 92:2099-2109)using the primers described supra. The results are shown in FIG. 3B. InFIG. 3B, panel a, lane S contains RNA from human kidney (Clontech, CA)which was used as a positive control since kidney is known toconstitutively synthesize high levels of OPN. Lanes 1-5 contain RNA fromautopsy samples of 5 representative control subjects without evidence ofcoronary artery disease. OPN-mRNA and GAPDH-mRNA are shown (panels a andb).

The results of RT-PCR using total RNA from control (FIG. 3B, panel a:lanes 1-5) and patient samples (FIG. 3B, panel b: lanes 1-5)corroborated the in situ hybridization results (FIG. 3A, panels a-d);while OPN-mRNA signal was virtually absent in control tissue,significant levels of OPN-mRNA were detected in tissue from coronaryatherectomy samples. The apparent lack of OPN-mRNA in control (autopsy)coronary arteries was not due to degradation of nucleic acids since thestrong RT-PCR amplification of mRNA of a house keeping gene, GAPDH, wasvirtually identical in each of these samples (FIG. 3B, bottom, panels a& b).

3. Immunofluorescence

In order to determine whether the outer (adventitia), middle (media) orthe inner (intima) tissue layers of the coronary arteries expressed OPNand α_(v)β₃ integrin, immunofluorescence was performed on bothDCA-patient and control tissues using antibodies against OPN(Chacklaparampil et al., (1996) Oncogene 12:1457-1467), humanSMC-specific α-actin monoclonal antibody (clone 1A4) (Sigma), and humanα_(v)β₃ integrin (Chemicon). Both SMC-specific α-actin and α_(v)β₃integrin were readily detectable in both patients and controls, and theintensity of SMC-specific α-actin and α_(v)β₃ integrin was also verysimilar in both controls and patients. In contrast, patient tissuesproduced a high level of OPN-specific immunofluorescence, whileOPN-immunofluorescence was virtually undetectable in the controltissues.

These results demonstrate that control and atherosclerotic tissuesexpress α_(v)β₃ integrin protein and that OPN protein levels areelevated in atherosclerotic tissue. Furthermore, these data also showthat expression of OPN and α_(v)β₃ integrin is specific to the smoothmuscle layer of coronary arteries of both control and atheroscleroticpatients.

4. Western Blotting

Western blot analysis was performed as described supra in Example 1, andthe results are shown in FIG. 3C. In FIG. 3C, panel a shows autopsysamples from 5 apparently normal coronary arteries (lanes 1-5). Panel bshows samples from 5 atherectomy patients (lanes 1-5). Lane S in panelsa and b contains purified OPN prepared from human milk as previouslydescribed [Senger et al. (1996) supra] which was used as a standard. Theresults of the Western blot analysis showed a virtual lack of OPN incontrol tissues (panel a: lanes 1-5) compared to the detection ofappreciable levels of OPN in atherectomy samples (panel b: lanes 1-5).The SDS-PAGE and Western blotting of patient tissue extracts revealedtwo distinct OPN bands as noted above (see FIG. 1A).

B. Plasma OPN Levels are Dramatically Elevated Following Angioplasty

Since significantly elevated OPN levels were detected in atherosclerotictissue as shown supra, and since OPN is a secreted protein, the effectof DCA on OPN levels in blood plasma was investigated. This was achievedby Western blot analysis of blood samples collected from DCA patients onthe day before the procedure, 24 h after, and at weekly intervals for 4weeks following DCA. Plasma samples which were prepared for OPNdetection as previously described (Senger et al., (1988) Cancer Res.48:5770-5774) were used for Western blotting using the method describedin Example 1, supra. Equal amounts of total plasma proteins were loadedin each lane for electrophoresis. Semi-quantitative, densitometricanalysis of the OPN bands in Western blots was performed using an LKBUltrascan LX-800 densitometer. Plasma samples from healthy individuals,who had no clinical evidence of coronary artery disease, served ascontrols. The results of the Western blot analysis are shown in FIG. 4.

In FIG. 4A, panel a, lanes 1-5 contain plasma samples from 5 differentcontrol patients containing equal amounts of protein as determined byspectrophotometric determination; lane S contains purified OPN standard.FIG. 4A, panel b, lanes 1-6 contain plasma samples from DCA patientsobtained 24 h before the procedure; panel c, lanes 1-6 contain plasmasamples from DCA patients 24 h after the procedure; panel d containsplasma samples from DCA patients obtained 3 weeks after DCA.

The results in FIG. 4A showed a dramatic difference in the levels ofplasma OPN between controls and DCA patients. Importantly, as shown inpanel a, the control plasma samples had virtually undetectable levels ofOPN (lanes 1-5), whereas, those from DCA patients, collected 24 h beforethe procedure (P, panel b), had readily visible OPN bands (lanes 1-6).Significantly, plasma OPN levels dramatically increased 24 h after DCA(panel c) and remained elevated even 3 weeks after the procedure (paneld).

Data obtained from a followup of relative densities of OPN bands,resolved by SDS-PAGE and Western blotting of plasma samples of threerepresentative DCA patients, collected over a 4 week period, is shown inFIG. 4B. In FIG. 4B, plasma samples were obtained from controls (C₁, C₂,and C₃) and patients 24 h before DCA (P). The numbers indicate the timein weeks after the procedure.

Surprisingly, the baseline plasma OPN levels of the patients, evenbefore the procedure, were remarkably higher than those of the healthycontrols. Moreover, plasma OPN levels showed a significant increasewithin 24 h following DCA, and these elevated plasma OPN levels weresustained for at least 4 weeks after DCA.

Taken together, the data unambiguously demonstrate (a) expression ofα_(v)β₃ integrin protein in CASMCs in the arteries of control andatherosclerotic patients [as detected by immunofluorescence], (b)remarkable elevation in the expression of OPN-mRNA [as detected by insitu hybridization and RT-PCR] and OPN protein [as detected by visualinspection and densitometric analysis of Western blots] in CASMCs in thearteries of patients suffering from coronary atherosclerosis as comparedto healthy individuals, (c) remarkable sustained elevation of OPNprotein levels in the serum of arterial atherosclerotic patientsfollowing DCA procedure as compared to the levels in healthy controlsand to atherosclerotic patients who did not undergo DCA.

Example 4 In Vitro Lipofection of Human Coronary Artery Smooth MuscleCells with Antisense OPN Sequences

In order to determine the efficacy of antisense OPN oligonucleotides inthe treatment of restenosis, antisense oligonucleotides designed to bindto mRNA encoded by the human OPN gene sequence were synthesized asphosphorothioate-oligonucleotides and their effect in vitro on themigration and proliferation of human coronary arterial smooth musclecells, and on the expression of OPN in these cells was determined.

A. Design and Synthesis of Antisense OPN Sequences

Five antisense OPN sequences were designed to bind to sequences withinthe coding region of the human OPN gene sequence depicted in FIG. 5 asfollows: ASHOPN-P1: 5′-AATCACTGCAATTCTCATGG-3′ (SEQ ID NO:9), ASHOPN-P2:5′-TTAACTGGTATGGCACAGGT-3′ (SEQ ID NO:10); ASHOPN-P3:5′-AGAACTTCCAGAATCAGCCT-3′ (SEQ ID NO:11); ASHOPN-P4:5′-TCGTTGGACTTACTTGGAAG-3′ (SEQ ID NO:12); and ASHOPN-P5:5′-ATGCTCATTGCTCTCATCAT-3′ (SEQ ID NO:13). For each of the antisensesequences, a corresponding control sense sequences was also designed.For the antisense ASHOPN-P1, the corresponding control sense sequencewas SHOPN-P6: 5′CCATGAGAATTGCAGTGATT-3′ (SEQ ID NO:14). The antisenseand sense sequences were synthesized asphosphorothioate-oligonucleotides by GIBCO-BRL (Life Technologies),Gaithersburg, Md.

B. In Vitro Lipofection

Human coronary artery smooth muscle cells (CASMCs) (Clonetics) weresubjected to lipofection with the antisense sequence ASHOPN-P1 or withthe control sense sequence SHOPN-P6, and the effect of lipofection wasmeasured on the proliferation of the lipofected cells and on theexpression of OPN as measured by Western blot analysis.

CASMCs (Clonetics) were transfected with a “LIPOFECTIN”-oligonucleotidecomplex according to the manufacturer's (GIBCO-BRL Life Technologies,Inc. Gaithersburg, Md.) specifications. Briefly, 5 μg of “LIPOFECTIN”was mixed with either 1 or 2 μg of antisense oligonucleotide in 200 μlof serum free medium (SFM) (also called basal medium) and incubated atroom temperature for 15 min. Cells (1×10⁵ cells/well, 12-well plate)during log phase of growth were washed with SFM and 1 ml of SFMcontaining Lipofectin with different amounts of antisenseS-oligonucleotides was added to each well and mixed by gentle agitation.The cells were incubated further with the same medium at 37° C. for 12h. Additional control cells which received either SFM, or SFM whichcontained “LIPOFECTIN” alone, were also included. At the end of the 12 hincubation period, cell viability was detected by trypan blue dyeexclusion test which showed the cells were healthy. The SFM mediumcontaining “LIPOFECTIN”-oligonucleotide complex was removed, and thecells incubated for an additional 48 h in regular medium. At the end ofthis incubation period, the cells were ready for the determination of adose response on OPN expression as measured by Western blotting.

C. Effect of Lipofection with Antisense OPN Sequences on Migration,Invasion of ECM, Proliferation and OPN Expression

The effect of antisense ASHOPN-P1 on the proliferation of human coronaryartery smooth muscle cells (CASMCs) was determined as described supra.Briefly, CASMCs which had been transfected with “LIPOFECTIN” alone, orin the presence of either OPN sense or OPN antisense oligonucleotides (1μg each) as described above were incubated further using PDGF-AB (100ng/ml) in the presence or absence of OPN (3 μg/ml) containing basalmedium (SFM) for 24 h. After 4 h, [3H]-thymidine (1 mCi/ml) was addedand the cells were maintained in culture for an additional 24 h underthe same culture conditions. The supernatant was removed and the cellswashed in basal medium (SFM) and lysed in 50% TCA. The acid precipitableradioactivity was measured using a scintillation beta counter (Beckman).The results of the proliferation assay are shown in FIG. 6.

FIG. 6 shows the effect of treatment of human CASMCs with “LIPOFECTIN”(5μg/200 μl) alone (control), or in the presence of sense sequence,SHOPN-P6 (1.5 μg/200 ml) or antisense sequence, ASHOPN-P1 (1.5 μg/200μl). The results in FIG. 6 show that treatment with the antisenseASHOPN-P1 sequence resulted in an 82% inhibition of cell proliferationas compared to cells treated with “LIPOFECTIN” alone, whereas cellstreated with the sense sequence, SHOPN, showed about 50% inhibition ofproliferation.

The effect of antisense OPN sequences on OPN expression was determinedby Western blot analysis as described above. FIG. 7 shows the expressionof OPN when cells were treated with “LIPOFECTIN” alone or two differentdoses of S-oligonucleotides antisense OPN sequences (lanes 1-3). Lane 1contains immunoprecipitates of cells treated with “LIPOFECTIN” alone;Lane 2 contains immunoprecipitates of cells treated with 1 μgS-oligonucleotide ASHOPN-P1 (ASHOPN); and Lane 3 containsimmunoprecipitates of cells treated with 2 μg S-oligonucleotideASHOPN-P1 (ASHOPN).

The results demonstrate that the antisense sequence ASHOPN-P1 wascapable of inhibiting expression of OPN by CASMCs and resulted in theinhibition of CASMC proliferation and OPN protein expression as comparedto either “LIPOFECTIN”-treated cells or to OPN sense-treated cells.Moreover, these results demonstrate that the effect of antisensesequence ASHOPN-P1 is specific.

Example 5

Testing Antisense OPN Sequences in a Rat Carotid Artery In Vivo ModelSystem

The effect of antisense OPN sequences on restenosis is investigated inan art-accepted rat carotid artery in vivo model by local administrationof antisense OPN sequences to arteries which had been traumatized bycatheterization, followed by the assessment of the effect of treatmenton OPN expression and restenosis.

A. Administration of Antisense OPN to Traumatized Rat Carotid Artery

Antisense OPN sequences are administered to traumatized rat carotidarteries via lipofection or as part of a pluronic gel. Traumatization ofthe rat carotid artery is an art-accepted method for investigatingrestenosis [Lee et al. (1993) Circulation Research 73:797-807; von derLeyen et al. (1994) FASEB J. 8:A802; Simons et al. (1992) Nature359:67-70 (1992); Edelman et al. (1992) J. Clin,. Invest. 89:465-473;Morishita et al. (1993) Proc. Natl. Acad. Sci. USA 90:8474-8478].

1. Lipofection

In order to traumatize rat arteries, the adventitia of the carotidartery are stripped as previously described [Simons et al. (1992) Nature359:67-70 (1992); Edelman et al. (1992) J. Clin,. Invest. 89:465-473;Morishita et al. (1993) Proc. Natl. Acad. Sci. USA 90:8474-8478] bysubjecting the left common carotid arteries of rats to balloonangioplasty which denudes endothelium and induces a highly reproducibleintimal migration/proliferation of SMCs over the entire length of theaffected blood vessel. Briefly, male Sprague-Dawley rats (average weight500 g) (Charles Rivers) are anaesthetized with Nembutal (4 mg per 100g), and the left carotid artery of each animal is isolated by a midlinecervical incision, suspended on ties and stripped of adventitia. A 2FFogarty catheter is introduced through the external carotid artery ofeach rat, advanced to the aortic arch, the balloon is inflated toproduce moderate resistance to catheter movement and then graduallywithdrawn to the entry point. The entire procedure is repeated threetimes for each animal. After vascular injury to the carotid artery, thedistal injured segment is transiently isolated by temporary ligatures.The oligonucleotide-“LIPOFECTIN” complex (prepared as described supra)is infused into the segment and incubated for 15 min at roomtemperature. After a 15-min incubation, the infusion cannula is removed,and blood flow to the carotid artery is restored by release of theligatures. Controls receive either “LIPOFECTIN” alone, or a complex of acorresponding sense oligonucleotide-“LIPOFECTIN”.

2. Pluronic Gel

After vascular injury of the carotid artery, the antisense OPNoligonucleotide sequences are added at a concentration of 1 mg ml⁻¹ to25% (w/v) solutions of F127 pluronic gels prepared following themanufacturer's (BASF Wyandotte Corporation) instructions, and maintainedat 4° C. Prechilled pipettes and tips are used to apply a 200 μlsolution to the carotid artery from which the adventitia is stripped. Oncontact with tissues at 39° C., the solution gels instantaneouslygenerating a translucent layer that envelops the treated region. Thewounds are closed immediately after application of the gel, and the ratsare returned to their cages. Inspection of additional animals isexpected to reveal that pluronic gel disappears over 1-2 h. Controlsreceive either pluronic gel alone, or pluronic gel containing acorresponding sense sequence.

B. Effect of Treatment with Antisense OPN on OPN Expression

The effect of antisense treatment on expression of OPN mRNA isdetermined by Northern analysis of the expression of thepreviously-described rat osteopontin cDNA sequence (Oldberg et al.(1986) Proc. Natl. Acad. Sci. USA 83:8819-8823) (SEQ ID NO:16) shown inFIG. 8.

The effect of antisense OPN oligonucleotides in suppressing OPN mRNAlevels in the rat carotid artery is investigated 2 weeks after injurywhen the extent of SMC accumulation has reached a maximum. The treatedportion of the blood vessel is surgically removed from five pairs ofantisense- and sense-treated rats. It is expected that injured carotidartery treated with antisense oligonucleotide exhibits lowered, orundetectable, levels of OPN mRNA as compared to injured carotid arterytreated with sense oligonucleotide.

The effect of antisense treatment on expression of OPN protein is alsodetermined by Western blot analysis according to methods known in theart (Singh et al. (1990) J. Biol. Chem. 265:18696-18701; Chakalaparampilet al. (1996) Oncogene 12:1457-1467). For Western blot analysis, thetreated portion of the blood vessel is surgically removed, and preparedfor Western blotting as described above, with the exception thatantibody which recognizes rat osteopontin, rather than humanosteopontin, is used. Antibodies which are cross-reactive with ratosteopontin include the previously-described anti-OPN serum (OST-1) aswell as the commercially available anti-fibronectin serum (CollaborativeResearch). OST-1 was raised against the synthetic oligopeptideNH₂-DPKSKEDDRYLKFRIS-COOH (SEQ ID NO:18), which represents amino acidresidues 291-306 of rat OPN (Singh et al. (1990) J. Biol. Chem.265:1869-1870). OST-1 is cross-reactive with both mouse and human OPNs(Singh el al. (1992) J. Biol. Chem. 267:2384-2385). Forimmunoprecipitation of proteins, aliquots containing equal amounts oftrichloroacetic acid-precipitated protein is diluted with 1 volume ofRIPA buffer (0.05 M Tris-HCl (pH 7.2), 0.15 M NaCl, 1% Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 100 Kalikrein inactivating units ofaprotinin/ml, 5 mM PMSF and 5 μg/ml of trypsin inhibitor) and incubatedat 4° C. for 2 h with 15 μl of anti-fibronectin or 25 μl of OST-1. Theresulting immune complexes are collected by adding an excess (30 μl of50% slurry in RIPA buffer) of protein A-Sepharose (PharmaciaBiotechnology, Inc.) to the reaction mixture and incubating for 1 h at4° C. with gentle agitation. The adsorbed immune complexes are pelletedby centrifugation, washed three times with RIPA buffer, twice with PBS,and finally rinsed with distilled water. The immunoprecipitatedproteins, s are subsequently suspended in 50 μl of sample buffer (0.07 MTris-HCl (pH 6.8), 3% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.01%bromophenol blue). To denature osteopontin-fibronectin complexes,samples readjusted to 0.2% SDS, incubated at 95° C. for 5 min, dilutedwith 1 volume of RIPA buffer lacking SDS, and immunoprecipitated witheither OST-1 or anti-fibronectin serum. Electrophoretic analysis bySDS-PAGE is then carried out on 10% slab gels.

It is expected that the levels of OPN in injured vessels treated withantisense will be reduced as compared with OPN levels in injured vesselswhich have received no treatment or which are treated with carrier (Le.,“LIPOFECTIN” or pluronic gel) alone or with a senseoligonucleotide/carrier complex.

C. Effect of Treatment with Antisense OPN on Restenosis

The effect of antisense OPN oligonucleotides on restenosis is determinedby measurement of vascular DNA synthesis and content, and of the effecton neointimal size.

1. Measurement of DNA Synthesis

For bromodeoxyuridine (BrdUrd) staining, BrdUrd is injected into ratsafter vascular injury (100 mg/kg subcutaneously and 30 mg/kgIntraperitoneally at 18 h prior to sacrifice and then 30 mg/kgintraperitoneally at 12 h prior to sacrifice). Rats are sacrificed onday 4 after the surgical procedure. The carotid artery is removed afterperfusion-fixation (110 mmHg; 1 mmHg=133 Pa) with 4% (wt/vol)paraformaldehyde and processed for immunohistochemistry by usinganti-BrdUrd antibodies (Amersham). The proportion of BrdUrd-positivecells is determined by cell counts under light microscopy in a blindedfashion. Measurement of DNA is performed at 4 days after the surgicalprocedure using bisbenzimide trihydrochloride (Pierce). It is expectedthat antisense treatment of injured arteries will inhibit BrdUrdincorporation (a marker of DNA synthesis and cell proliferation) in thevessel wall as compared to the sense-treated controls or to theuntreated injured control vessels.

2. Morphometric Analysis

Formation of neointima along the length of the treated artery isdetermined at 2, 4, and 8 weeks after the surgical procedure. At thetime of killing, the animals are anaesthetized with Nembutal andperfused with 150 cc normal saline under a pressure of 120 mm Hg. Thecarotid arteries are removed, fixed in 3% formalin, and processed forlight microscopy in a standard manner. Three individual sections fromthe middle of surgically treated segments which are treated withantisense sequences are analyzed by measuring the mean cross-sectionalareas of the intimal and of the medial regions which are untreated,treated with carrier (i.e., pluronic gel or “LIPOFECTIN”) alone, treatedwith carrier plus antisense oligonucleotide, or treated with carrierplus sense oligonucleotide. These measurements are used to determine aratio of intimal to medial cross-sectional areas. In addition, threesections from the middle section of the injured region which has notreceived antisense treatment are also analyzed. Animals are coded sothat operation and analysis are performed without knowledge of whichtreatment individual animals receive. It is expected that treatment withantisense will result in a reduction of the cross-sectional ratio ofintima/media as compared with the cross-sectional ratio of intima/mediain control injured arteries receiving no treatment, carrier alone, or acarrier/sense oligonucleotide complex.

In order to determine the selectivity of the antisense effect, a doseresponse (e.g., 1 μM-20 μM antisense) of the effect on theintimal/medial cross-sectional ratio is determined at the site ofoligonucleotide administration. Additionally, the selectivity of theantisense effect is determined by measuring the intimal/medialcross-sectional ratio along the length of the treated vessel in whichthe site of oligonucleotide administration is marked with silk ties. Itis expected that the intimal/medial cross-sectional ratio in injuredvessels treated with antisense will be reduced as compared with theratio in injured vessels which have received no treatment or which aretreated with carrier (i.e. “LIPOFECTIN” or pluronic gel) alone or with asense oligonucleotide/carrier complex. Such a reduction in ratioindicates that the antisense molecule is useful in reducing restenosisin a human subject.

As clear from the data presented herein, the present invention has theadvantage of providing methods and compositions for preventing and/ortreating restenosis. In particular, the OPN antisense sequences areuseful in preventing the development of restenosis in angioplastyprocedures. Furthermore, OPN antisense sequences provide a tool forspecific therapy with minimal potential adverse side-effects in view ofthe ability of the sequences specifically to target expression of asingle gene which is implicated in the development of restenosis.Moreover, OPN antisense sequences as disclosed herein are easy toadminister and are effective over a short period of time.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artare intended to be within the scope of the following claims.

18 1 20 DNA Artificial Sequence Synthetic 1 ctacaaccag catatcttca 20 220 DNA Artificial Sequence Synthetic 2 caccagtctg atgagtctca 20 3 20 DNAArtificial Sequence Synthetic 3 tccatgtgtg aggtgatgtc 20 4 18 DNAArtificial Sequence Synthetic 4 ccatggagaa ggctgggg 18 5 20 DNAArtificial Sequence Synthetic 5 caaagttgtc atggatgacc 20 6 19 DNAArtificial Sequence Synthetic 6 ctaagcagtt ggtggtgca 19 7 5 PRTArtificial Sequence Synthetic 7 Gly Arg Gly Asp Ser 1 5 8 40 DNAArtificial Sequence Synthetic 8 tccatgtgtg aggtgatgtc ctcgtctgtagcatcagggt 40 9 20 DNA Homo sapiens 9 aatcactgca attctcatgg 20 10 20 DNAHomo sapiens 10 ttaactggta tggcacaggt 20 11 20 DNA Homo sapiens 11agaacttcca gaatcagcct 20 12 20 DNA Homo sapiens 12 tcgttggact tacttggaag20 13 20 DNA Homo sapiens 13 atgctcattg ctctcatcat 20 14 20 DNA Homosapiens 14 ccatgagaat tgcagtgatt 20 15 1422 DNA Homo sapiens 15gaccagactc gtctcaggcc agttgcagcc ttctcagcca aacccgacca aggaaaactc 60actaccatga gaattgcagt gatttgcttt tgcctcctag gcatcacctg tgccatacca 120gttaaacagg ctgattctgg aagttctgag gaaaagcagc tttacaacaa atacccagat 180gctgtggcca catggctaaa ccctgaccca tctcagaagc agaatctcct agccccacag 240aatgctgtgt cctctgaaga aaccaatgac tttaaacaag agacccttcc aagtaagtcc 300aacgaaagcc atgaccacat ggatgatatg gatgatgaag atgatgatga ccatgtggac 360agccaggact ccattgactc gaacgactct gatgatgtag atgacactga tgattctcac 420cagtctgatg agtctcacca ttctgatgaa tctgatgaac tggtcactga ttttcccacg 480gacctgccag caaccgaagt tttcactcca gttgtcccca cagtagacac atatgatggc 540cgaggtgata gtgtggttta tggactgagg tcaaaatcta agaagtttcg cagacctgac 600atccagtacc ctgatgctac agacgaggac atcacctcac acatggaaag cgaggagttg 660aatggtgcat acaaggccat ccccgttgcc caggacctga acgcgccttc tgattgggac 720agccgtggga aggacagtta tgaaacgagt cagctggatg accagagtgc tgaaacccac 780agccacaagc agtccagatt atataagcgg aaagccaatg atgagagcaa tgagcattcc 840gatgtgattg atagtcagga actttccaaa gtcagccgtg aattccacag ccatgaattt 900cacagccatg aagatatgct ggttgtagac cccaaaagta aggaagaaga taaacacctg 960aaatttcgta tttctcatga attagatagt gcatcttctg aggtcaatta aaaggagaaa 1020aaatacaatt tctcactttg catttagtca aaagaaaaaa tgctttatag caaaatgaaa 1080gagaacatga aatgctcttt ctcagtttat tggttgaatg tgtatctatt tgagtctgga 1140aataactaat gtgtttgata attagtttag tttgtggctt catggaaact ccctgtaaac 1200taaaagcttc agggttatgt ctatgttcat tctatagaag aaatgcaaac tatcactgta 1260ttttaatatt tgttattctc tcatgaatag aaatttatgt agaagcaaac aaaatacttt 1320tacccactta aaaagagaat ataacatttt atgtcactat aatcttttgt tttttaagtt 1380agtgtatatt ttgttgtgat tatctttttg tggtgtgaat aa 1422 16 1473 DNA RattusNorvegicus CDS (80)..(1030) 16 gcaagcctca gcatccttgg ctttgcagtctcctgcggca agcattctcg aggaagccag 60 ccaaggacca actacaacc atg aga ctg gcagtg gtt tgc ctt tgc ctg ttc 112 Met Arg Leu Ala Val Val Cys Leu Cys LeuPhe 1 5 10 ggc ctt gcc tcc tgt ctc ccg gtg aaa gtg gct gag ttt ggc agctca 160 Gly Leu Ala Ser Cys Leu Pro Val Lys Val Ala Glu Phe Gly Ser Ser15 20 25 gag gag aag gcg cat tac agc aaa cac tca gat gct gta gcc act tgg208 Glu Glu Lys Ala His Tyr Ser Lys His Ser Asp Ala Val Ala Thr Trp 3035 40 ctg aag cct gac cca tct cag aag cag aat ctt cta gcc cca cag aat256 Leu Lys Pro Asp Pro Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn 4550 55 tct gtg tcc tct gaa gaa acg gat gac ttt aag caa gaa act ctt cca304 Ser Val Ser Ser Glu Glu Thr Asp Asp Phe Lys Gln Glu Thr Leu Pro 6065 70 75 agc aac tcc aat gaa agc cat gac cac atg gac gat gat gac gac gac352 Ser Asn Ser Asn Glu Ser His Asp His Met Asp Asp Asp Asp Asp Asp 8085 90 gat gac gac gga gac cat gca gag agc gag gat tct gtg aac tcg gat400 Asp Asp Asp Gly Asp His Ala Glu Ser Glu Asp Ser Val Asn Ser Asp 95100 105 gaa tct gac gaa tct cac cat tcc gat gaa tct gat gag tcc ttc act448 Glu Ser Asp Glu Ser His His Ser Asp Glu Ser Asp Glu Ser Phe Thr 110115 120 gcc agc aca caa gca gac gtt ttg act cca atc gcc ccc aca gtc gat496 Ala Ser Thr Gln Ala Asp Val Leu Thr Pro Ile Ala Pro Thr Val Asp 125130 135 gtc cct gac ggc cga ggt gat agc ttg gct tac gga ctg agg tca aag544 Val Pro Asp Gly Arg Gly Asp Ser Leu Ala Tyr Gly Leu Arg Ser Lys 140145 150 155 tcc agg agt ttc cct gtt tct gat gaa cag tat ccc gat gcc acagat 592 Ser Arg Ser Phe Pro Val Ser Asp Glu Gln Tyr Pro Asp Ala Thr Asp160 165 170 gag gac ctc acc tcc cgc atg aag agc cag gag tcc gat gag gctatc 640 Glu Asp Leu Thr Ser Arg Met Lys Ser Gln Glu Ser Asp Glu Ala Ile175 180 185 aag gtc atc cca gtt gcc cag cgt ctg agc gtg ccc tct gat caggac 688 Lys Val Ile Pro Val Ala Gln Arg Leu Ser Val Pro Ser Asp Gln Asp190 195 200 agc aac ggg aag acc agc cat gag tca agt cag ctg gat gaa ccaagc 736 Ser Asn Gly Lys Thr Ser His Glu Ser Ser Gln Leu Asp Glu Pro Ser205 210 215 gtg gaa aca cac agc ctg gag cag tcc aag gag tat aag cag agggcc 784 Val Glu Thr His Ser Leu Glu Gln Ser Lys Glu Tyr Lys Gln Arg Ala220 225 230 235 agc cac gag agc act gag cag tcg gat gcg atc gat agt gccgag aag 832 Ser His Glu Ser Thr Glu Gln Ser Asp Ala Ile Asp Ser Ala GluLys 240 245 250 ccg gat gca atc gat agt gca gag cgg tcg gat gct atc gacagt cag 880 Pro Asp Ala Ile Asp Ser Ala Glu Arg Ser Asp Ala Ile Asp SerGln 255 260 265 gcg agt tcc aaa gcc agc ctg gaa cat cag agc cac gag tttcac agc 928 Ala Ser Ser Lys Ala Ser Leu Glu His Gln Ser His Glu Phe HisSer 270 275 280 cat gag gac aag cta gtc cta gac cct aag agt aag gaa gatgat agg 976 His Glu Asp Lys Leu Val Leu Asp Pro Lys Ser Lys Glu Asp AspArg 285 290 295 tat ctg aaa ttc cgc att tct cat gaa tta gag agt tca tcttct gag 1024 Tyr Leu Lys Phe Arg Ile Ser His Glu Leu Glu Ser Ser Ser SerGlu 300 305 310 315 gtc aat taaagaagag gcaaaaccac agttccttac tttgctttaaataaaacaaa 1080 Val Asn aagtaaattc caacaagcag gaatactaac tgcttgtttctcagttcagt ggatacatgt 1140 atgtggacaa agaaatagat agtgttttgg gccctgagcttagttcgttg tttcatgcag 1200 acaccactgt aacctagaag tttcagcatt tcgcttctgttctttctgtg caagaaatgc 1260 aaatggccac tgcattttaa tgattgctat tcttttatgaataaaatgta tgtagaggca 1320 ggcaaactta caggaacagc aaaattaaaa gagaaactataatagtctgt gtcactataa 1380 tcttttggtt ttataattag tgtatatttt gttgtgattatttttgttgg tgtgaataaa 1440 tcttgtatct tgaatgtaaa aaaaaaaaaa aaa 1473 17317 PRT Rattus Norvegicus 17 Met Arg Leu Ala Val Val Cys Leu Cys Leu PheGly Leu Ala Ser Cys 1 5 10 15 Leu Pro Val Lys Val Ala Glu Phe Gly SerSer Glu Glu Lys Ala His 20 25 30 Tyr Ser Lys His Ser Asp Ala Val Ala ThrTrp Leu Lys Pro Asp Pro 35 40 45 Ser Gln Lys Gln Asn Leu Leu Ala Pro GlnAsn Ser Val Ser Ser Glu 50 55 60 Glu Thr Asp Asp Phe Lys Gln Glu Thr LeuPro Ser Asn Ser Asn Glu 65 70 75 80 Ser His Asp His Met Asp Asp Asp AspAsp Asp Asp Asp Asp Gly Asp 85 90 95 His Ala Glu Ser Glu Asp Ser Val AsnSer Asp Glu Ser Asp Glu Ser 100 105 110 His His Ser Asp Glu Ser Asp GluSer Phe Thr Ala Ser Thr Gln Ala 115 120 125 Asp Val Leu Thr Pro Ile AlaPro Thr Val Asp Val Pro Asp Gly Arg 130 135 140 Gly Asp Ser Leu Ala TyrGly Leu Arg Ser Lys Ser Arg Ser Phe Pro 145 150 155 160 Val Ser Asp GluGln Tyr Pro Asp Ala Thr Asp Glu Asp Leu Thr Ser 165 170 175 Arg Met LysSer Gln Glu Ser Asp Glu Ala Ile Lys Val Ile Pro Val 180 185 190 Ala GlnArg Leu Ser Val Pro Ser Asp Gln Asp Ser Asn Gly Lys Thr 195 200 205 SerHis Glu Ser Ser Gln Leu Asp Glu Pro Ser Val Glu Thr His Ser 210 215 220Leu Glu Gln Ser Lys Glu Tyr Lys Gln Arg Ala Ser His Glu Ser Thr 225 230235 240 Glu Gln Ser Asp Ala Ile Asp Ser Ala Glu Lys Pro Asp Ala Ile Asp245 250 255 Ser Ala Glu Arg Ser Asp Ala Ile Asp Ser Gln Ala Ser Ser LysAla 260 265 270 Ser Leu Glu His Gln Ser His Glu Phe His Ser His Glu AspLys Leu 275 280 285 Val Leu Asp Pro Lys Ser Lys Glu Asp Asp Arg Tyr LeuLys Phe Arg 290 295 300 Ile Ser His Glu Leu Glu Ser Ser Ser Ser Glu ValAsn 305 310 315 18 16 PRT Artificial Sequence Synthetic 18 Asp Pro LysSer Lys Glu Asp Asp Arg Tyr Leu Lys Phe Arg Ile Ser 1 5 10 15

What is claimed is:
 1. A method of diminishing osteopontin expression,comprising: a) providing: i) a human smooth muscle cell in culture; andii) an osteopontin antisense oligonucleotide complementary to apolynucleotide selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; and b)administering an amount of said oligonucleotide to said smooth musclecell in culture under conditions such that said osteopontin expressionis diminished.
 2. The method of claim 1, wherein said human smoothmuscle cell is a coronary artery smooth muscle cell.
 3. The method ofclaim 1, wherein said osteopontin antisense oligonucleotide comprisesone or more phosphorothioate linkages.
 4. The method of claim 1, whereinsaid osteopontin antisense oligonucleotide is entrapped in a liposome.5. A method of reducing human smooth muscle cell proliferation,comprising: a) providing: i) a human smooth muscle cell in culture; andii) an osteopontin antisense oligonucleotide complementary to apolynucleotide selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; and b)administering an amount of said oligonucleotide to said human smoothmuscle cell under conditions such that proliferation of said humansmooth muscle cell is diminished.
 6. The method of claim 5, wherein saidosteopontin antisense oligonucleotide comprises one or morephosphorothioate linkages.
 7. The method of claim 6, wherein saidosteopontin antisense oligonucleotide is entrapped in a liposome.